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245ANALESRANFwwwanalesranfcomBiotininterferenceinseveralelectrochemiluminescenceimmunoassays(eclia)JoséLuisMartín-CalderónAnRealAcadFarmVol87nº3(2021)·pp239-246Pharmacokinetics studiesperformed withbiotininhealthyvolunteersreceivingseveraloraldosesrevealthatbiotinisrapidlyabsorbedandhasan effective serumhalf-life of15h(14)Inagroupreceivingasingleoraldoseof12mgpeakserumbiotinlevelsreached55ng/mL20Inthegroupsthatreceivedasingledoseof100or 300mgpeakserumbiotinreached 4949±161ng/mL(at125h)and8238± 3031ng/mL(at15h)respectively(21) Serumbiotinlevels 48hafteradministrationoforalbiotinat variousdoseswere71-439ng/mL(5mgofbiotin)106-568ng/mL(10mgofbiotin)169-690ng/mL(100mgbiotin)and669-1160ng/mL(300mg ofbiotin)(7) Theresults ofpopulationpharmacokineticanalysisshowthataftertheadministrationofdailydosesofbiotinofupto1mg(80-fold theadequatedailyintake) whichare foundin standardsupplement/multivitaminpillsserumbiotinlevelsfallbelow10ng/mLafter2hoursFordosesofbiotinofupto10mg/day(morethan300-foldtheadequatedailyintakeofbiotin)athresholdofserumbiotinlevelof30ng/mLwasreachedafter8hPharmacokinetics studies suggest thata washoutperiod ofbiotinisdesirablebeforeperformingbloodanalyseswhicharesusceptible tobiotininterferenceForassays withaninterference threshold of 30ng/mLan8h washoutperiodis enough tomitigatebiotininterferenceinpatients takingbiotindosesofup10mg(14)However,biotininterferenceinlaboratorytestresultspersistedforupto24hinpatientstakinghighdosesofbiotin(30mg)(22)Consequently,manymanufacturesrecommenddiscontinuingbiotinintakeforatleast24hbeforebloodsampling(7)Bothimmunoassaymanufacturersand clinicallaboratoriesaremakingefforts topreventerrorsdue tobiotininterferenceManufacturershaveincluded the threshold ofbiotininterferencein theirpackageinsertsClinicalbiochemistsmustwarnthecliniciansaboutbiotininterferencemedicalimpactSomemanufacturersaredevelopingmethodstominimizebiotininterferencesuchastheuseofstreptavidinbeadstocaptureexcessbiotin(18)Alimitationofour studyis that wehavenot consideredinterferencesdue tobiotinrelatedmetabolites suchasnorbiotin orbiotin sulfoxide(20)because thebinding ofbiotinderivatives toavidinhasbeendescribedalthoughthisbindingislesstightthanthatoftheparentalcompound5CONCLUSIONSInsummary,apartfromtheCA153andTSHassaysalltheothermethodstestedinourstudyweresusceptibletobiotininterferencewithintheexaminedbiotinconcentrationrangeFalselylow valuesoccurredinsandwichassaysandhighbiasincompetitiveassaysCliniciansandlaboratoriansshouldbeawareofthemedicalimportanceofbiotininterferenceasacauseofmisdiagnosisandincorrecttreatment4DISCUSSIONIn our study wehave analyzed the vulnerability of theresultsofelectrochemiluminescenceimmunoassaysontheRochee801ande411platformswhenrisingconcentrationsofbiotinwereadded Theaddition ofbiotin to the samplesproduced falselylowresultsin sandwich assays(negativeinterference) and spuriouslyhighresultsin competitiveassays(positiveinterference)inagreementwithpreviousreportsIn some cases the effect ofbiotininterference doesnot dependonanalyteconcentrationHowever,forFSHLHprostatespecificantigenfreeprostatespecificantigenvitaminB1225-hydroxyvitaminDparathyrinecortisolandC-terminalcollagentypeIpeptidestheinterferencediddependonanalyteconcentrationBiotininterferencewasespeciallynoteworthyintheantiperoxidaseantibodies(aTPO)test which was affected even at thelowestbiotin concentration(3125ng/mL)On theoppositeCA153and TSH werenotaffectedatallbybiotinpresenceonthesampleElectrochemiluminescenceimmunoassayisusedinRocheplatformsIn the sandwich design one antibodyislabeled withruthenium whichproduces the signal and the second antibodyisbound tobiotin whichis the capturedmoiety(17) The antibody complexis capturedbystreptavidin-coatedparticlesTheanalyteconcentrationisproportional to the signalBiotin containedin the samplebinds to thestreptavidin-coatedparticlesallowingthecouplingoflessantibodycomplexes with thesemicroparticlesConsequently, theresults are falsely decreasedIn contrastin competitive assays the antigenislabeledwithrutheniumandcompeteswiththeanalyteforthebiotynilatedantibodiesIn this case theunlabeled analyte on the sample competeswithruthenium-labeledantigenforthebiotinylatedantibodiesandthemeasuredsignalisinverselyproportionaltotheconcentrationofanalyteThereforebiotinpresenceyieldsfalselyhighresultsTheincreaseduseofbiotinsupplementsandhighdosesoftherapeuticbiotinisnot only a specificproblem for electrochemiluminescenceimmunoassaysbutalsoachallenge forallclinicallaboratorieswhichemploystreptavidin-biotinbasedimmunoassays(171819)Indeedinvestigationsperformed with spiked samples and studiescarriedoutwithhealthyvolunteershaveshownthatbiotininterfereswithmanyhomogeneousandheterogeneousimmunoassayswhichrelyonthestreptavidin-biotinsystem(11)Biotin´sinterferenceisparticularlyimportantin thyroidhormone assays with severalreported cases ofmisdiagnosedGrave´s disease(71012) Moreover,biotin´sinterferencecanexplainotherunexpectedresultsinendocrineassayssuchashighcortisolemiasassociatedwithACTHsuppressionorelevatedconcentrationsof25-OHvitaminDassociatedwithlowPTHlevels(7)both due to thepresence ofbiotinin the sample causingfalselylowresultsinsandwichassays(proteinpeptides)butfalselyhighresultsincompetitiveassays(smallmolecules)